1.1 Field of the Invention
This invention relates to the purification of chemical substances, and, more particularly, to devices, methods, and systems for performing chemical purification and analysis. More particularly, the devices, methods, and systems provided by the invention have particularly useful application in the purification and analysis of nucleic acids, and, more particularly to microfluidic devices for performing such purification and analysis. The invention has applications in the areas of analytical chemistry, forensic chemistry, microfluidics and microscale devices, medicine, and public health.
1.2 The Related Art
The extension of semiconductor fabrication techniques to create highly miniaturized chemical devices (Beach, Strittmatter et al. 2007) has created a revolution in analytical chemistry, especially by providing a means for identifying chemical substances present in minute concentrations in complex mixtures with great precision and accuracy. This revolution has had noticeable impact in chemical processing, medicine, forensic science, and national defense, where such devices provide fast, portable, and economic biological detectors. Examples of such devices include devices for collecting and identifying particulates (Wick 2007), systems for detecting molecular contaminants (Knollenberg, Rodier et al. 2007), and devices for detecting proteins (Terry, Scudder et al. 2004; Deshmukh 2006). Other devices use fluidic technologies to isolate and/or amplify nucleic acids using Polymerase Chain Reaction (PCR) in an automated system. Examples of such devices are those sold commercially by Qiagen (Hilden, Germany), Roche (Basel, Switzerland), Applied Biosystems (Foster City, Calif.), Idaho Technologies (Salt Lake City, Utah), and Cepheid (Sunnyvale, Calif.).
But as with any analytical process, preparing the sample prior to processing is critical to good performance. The presence of too many complicating factors and concentrations of substances that may mask analytes of interest can render robust detection all but impossible. This problem is of particular concern when attempting to analyze the nucleic acid content of cell lysates, which are extremely complex and heterogenous mixtures (Colpan 2001). The preparatory task is made still more difficult where portable analytical devices are concerned, since those devices are expected to be used in locations where common laboratory support equipment, such as centrifuges and separation columns, are not available. In those cases, some means for filtering a raw sample, such as a blood or urine sample, is critical to providing meaningful results. Current devices based on fluidic technologies, in particular the above-mentioned Qiagen devices, use glass filters that are soft and compliant, requiring a support matrix. The filters have small pore sires, typically between about one- and three microns, to get efficient capture of the nucleic acids from the sample. Because of the small pores sires, the filters are also relatively thin, typically less than two millimeters thick to reduce fluid flow resistance when sample is forced though the small pore sires. In the Qiagen procedure, typically a sample is mixed with a chaotropic agent, such as guanidine, and the mixture is passed through the glass filter using centrifugal force, in which fluid flows in only one direction. Nucleic acids bind to the glass filter; they are washed with ethanol or isopropanol, and subsequently released using a ten millimolar (10 mM) Tris buffer at a pH of about eight (pH 8.0) or water. But the small pore sizes limit the amount of sample that can be processed, due to resistance created by fluid flow and potential for clogging created by greater flow rates. Thus, devices such as the Qiagen devices can be easily damaged or otherwise rendered ineffective easily. Moreover, these characteristics limit sample input volume, the types of samples that can be examined, large-volume samples, concentration factors, and simple fluidic integration.
Larger glass filters have been used to provide pre-processing filtration of samples. For example, U.S. Pat. No. 4,912,034 (Kalra, Pawlak et al. 1990) describes an immunoassay for detecting a target analyte in a liquid sample that includes an optional prefilter assembly made of glass fibers. However, this device is not a microfluidic device and does not show or suggest the use of glass frits as a filter prior to microscale PCR reactions. U.S. Pat. No. 4,923,978 (McCormack 1990) describes prior uses of glass fiber filters to remove unwanted protein- and protein-DNA complexes from aqueous DNA samples, but in a disparaging manner noting that such filters have low binding capacities (see Column 2). Indeed, the '978 patent claims a very different material for performing such filtrations. U.S. Pat. No. 6,274,371 (Colpan 2001) describes silica gel, aluminum oxide, and diatomaceous earth as a preferred filtering agent for removing unwanted contaminants from cellular lysates prior to nucleic acid analysis. U.S. Pat. No. 6,800,752 (Tittgen 2004) describes using a chromatography material to separate mixtures comprising nucleic acids, in which the material includes carrier and ion exchanger functions wherein the carrier comprises a fibrous material on a support, such as a plastic frit.
Nevertheless, there remains therefore a need to provide fluidic devices that are effective to isolate and identify nucleic acids that overcome the limitations of the current generation of such devices. The present invention meets these and other needs.